Tuesday, March 15, 2016

Guide RNAs: A Uniquely Kinetoplastid Way of Editing Mitochondrial mRNA

by Paul Hoffman

Mitochondria
are found nearly all eukaryotic cells on Earth, generating most of the energy
for eukaryotic life. These organelles have their own genome, independent from
the nuclear genome that controls a majority of a cell’s functions. Much like
nuclear DNA, mitochondrial DNA, or mtDNA, follows the central dogma of molecular
biology: DNA gets transcribed to messenger RNA, or mRNA, which gets translated
to protein or other products. The roles of the mitochondrial genome vary by
organism, with some species utilizing their mtDNA more than others. As such,
expression of mtDNA varies wildly across eukaryotic life. The kinetoplastids,
for example, are a class of protozoans known for having large mitochondria with
a massive network of mtDNA, containing multiple duplicates of the entire
mitochondrial genome. Kinteoplasts are organized into circular fragments, which
vary in size and function. Unlike most mtDNA, or nearly all DNA for that
matter, kinetoplast DNA, or kDNA, is encrypted and mRNA produced from it needs
to be decoded before it can be translated into useable products. A unique
editing process is required for the mRNA to be functional, and this is what
makes the kinetoplastids so unique.

Guide RNAs, or gRNAs, are encoded
by certain fragments of the kDNA. Kinetoplast fragments can be organized into
two classes: maxicircles, approximately twenty thousand to forty thousand
base-pairs in length, and minicircles, approximately five hundred to one
thousand base pairs in length. Maxicircles encode protein products, and are
transcribed into mRNA transcripts. These transcripts are called cryptogenes;
the message that is normally readable in regular mRNA is hidden due to highly
conserved mutations within kDNA. In order for the message to be read by the
cellular machinery, the mRNA transcripts need to be decoded into mRNA, and that
is where gRNAs come into play. gRNAs come from the minicircles, vary from
species to species (1), and range in number and
class between species (2).

There are two major subgroups
within the kinetoplastids, the trypanosomatids and the bononids, with major
differences to gRNAs and kinetoplast structure between, and even within, these
groups (3). Leishmania species fall under the former group, and form a clade,
or close relationship, with Crithidia species
within the trypanosomatids, with the Trypanosoma
species forming another (4). This clade only has one gRNA
coded per minicircle. Some trypanosomes contain multiple genes per minicircle (5), with three gRNAs per
minicircle being coded for in some species of Trypanosoma (6). RNA editing in the Trypanosoma clade is performed at both
ends of each editing domain while theLeishmania-Crithidia clade is at the 5’ prime end of a domain (3). The bononids are often seen
as more primitive than their cousins. Unlike the trypanosomatids, the bononids
have unconcatenated ktDNA, meaning that it is not tightly bound, but rather
loose and free-floating. Furthermore, the bononids have two gRNAs encoded per
minicircle, unlike the Leishmania-Crithidia clade’s one or the Trypanosoma’s three to four (3). These differences make the
kinetoplasts and gRNAs unique between the subdivisions of the kinetoplastids
and incompatible between one another, despite sharing a common lineage. Of
these groups, the trypanosomatids are far better studied, and seen as the model
for studying gRNAs.

Of the two
clades that exist in the trypanosomatids, the Leishmania-Crithidia clade is often less studied, but just as
important as these are the organisms with the most amount of classification to
gRNAs. In some species of Leishmania,
there can be upwards of sixty different classes of minicircles, each of which
can encode multiple gRNAs. Each class is a unique minicircle sequence that
codes for a group of gRNAs that act on a certain mRNA sequence (7). Minicircles all share a
conserved sequence and differentiate based on variable sequences (8). In Leishmania species, the coding region for gRNAs is around one
hundred fifty base-pairs from the conserved region; each minicircle codes for a
single specific gRNA (9). Certain Leishmania species contain gRNAs with duplicate functions, with
some having upwards of nineteen redundant gRNAs (10). These redundant gRNAs vary
in expression level, with some of the redundant gRNAs being completely
non-functional (5). Some of this redundancy may
allow for minicircles to be lost and gained without any major change in
function to the cell. Leishmania
species have a wide range of minicircle numbers, even between strains of the
same species, yet are able to function normally and have all the required gRNAs
needed for survival (3).

gRNA
function can be boiled down to adding or removing uracil-residues from mRNA
transcripts (11). All genetic material is
coded for with nucleotides: DNA uses adenine, guanine, cytosine, and thymine;
while RNAs swap out thymine for uracil. gRNAs decode mRNA transcripts by
shifting the coding region of these transcripts by the addition and deletion of
uracils in the transcript. The gRNAs have precise points within the transcripts
where they perform their editing: gRNAs bind to a block of bases, ranging from
one to ten nucleotides, within the transcript and signal a cleavage reaction in
these blocks (12). Multiple blocks are strung
together to create an editing domain. It is within these domains that the vast
majority of mRNA transcript editing occurs. Different combinations of gRNA
blocks can be formed on the same transcript to create different domains,
yielding different mRNA products from the same transcript. Should a transcript
be misedited by the wrong gRNA, other gRNAs can re-edit the transcript to form
a correct sequence (12).

A big
question is how gRNAs, and the kinetoplast structure as a whole, arose from the
standard mitochondrion. Evidence for early divergence ofthe kinetoplastids from the euglenoids comes
from ribosomal RNA sequences (13). The kinetoplastids are
highly divergent from other euglenoids and, unlike many other groups, are
monophyletic (14). The first models on the
origin of the kinetoplast and gRNAs is a three step process: the ability to
edit mRNA is acquired from enzymatic activity, then mutations occur at certain
sites within the mRNA, finally editing mRNA becomes essential for growth (15). Expansions to this model
propose that cryptogenes could then code for multiple proteins with different
editing procedures unlocking different protein products from the mRNA. Another
model suggests that RNA editing with gRNAs arose to combat mutations. kDNA,
like other mtDNA, is highly unstable when not in use. Leishmania species have complex lifecycles, and different genes
within the kDNA are needed at different times. When selective pressure is
relived from kDNA and mtDNA, mutation rates tend to skyrocket within the sequences,
leading to a vastly different kinetoplast genome than before. RNA editing with
gRNAs could combat these mutations, restoring lost functionality by returning
the mRNA transcripts to a working state. Furthermore, the kinetoplastids is
that they do not use the universal genetic code (3). In most organisms, the codon
UGA signals and end to reading an mRNA sequence when translating from nucleic
acids to protein. Kinetoplastids, however, use UGA to code for tryptophan. However,
the kinetoplasts themselves do not code for tRNA, or transfer RNA, that carries
tryptophan to the mRNA sequence in the process of translating to protein.
Because these tRNAs are imported into the kinetoplast, they rely on the genetic
code of the cell rather than that of the kinetoplast. The differences between
the cell’s code and the kinetoplast’s code may warrant RNA editing to be
preserved and selected for in the kinetoplastids.

Kinetoplasts
and gRNAs make up the unique mitochondria system in kinetoplastids. Unlike
nearly every other eukaryote, the kinetoplastids have the unique capability to
change the expression of their mitochondrial genes by changing their mRNA
transcripts using other RNAs as the editor. While the origin of gRNAs are still
unclear, their function is well classified and incredibly interesting as no
other organism uses this mechanism of RNA editing.

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